Haotian Zhao

Post-transcriptional down-regulation of Atoh1/Math1 by bone morphogenic proteins suppresses medulloblastoma development

Bone morphogenic proteins 2 and 4 (BMP2 and BMP4) inhibit proliferation and induce differentiation of cerebellar granule neuron progenitors (GNPs) and primary GNP-like medulloblastoma (MB) cells. This occurs through rapid proteasome-mediated degradation of Math1 (Atoh1), a transcription factor expressed in proliferating GNPs. Ectopic expression of Atoh1, but not of Sonic hedgehog (Shh)-regulated Gli1 or Mycn, cancels these BMP-mediated effects and restores Shh-dependent proliferation of GNPs and MB cells in vitro and in vivo. Genes regulating the BMP signaling pathway are down-regulated in mouse MBs. Thus, BMPs are potent inhibitors of MB and should be considered as novel therapeutic agents.

Atoh1 Inhibits Neuronal Differentiation and Collaborates with Gli1 to Generate Medulloblastoma-Initiating Cells

The morphogen and mitogen Sonic Hedgehog (Shh) activates a Gli1-dependent transcription program that drives proliferation of granule neuron progenitors (GNP) within the external germinal layer of the postnatally developing cerebellum. Medulloblastomas with mutations activating the Shh signaling pathway preferentially arise within the external germinal layer, and the tumor cells closely resemble GNPs. Atoh1/Math1, a basic helix-loop-helix transcription factor essential for GNP histogenesis, does not induce medulloblastomas when expressed in primary mouse GNPs that are explanted from the early postnatal cerebellum and transplanted back into the brains of naïve mice. However, enforced expression of Atoh1 in primary GNPs enhances the oncogenicity of cells overexpressing Gli1 by almost three orders of magnitude. Unlike Gli1, Atoh1 cannot support GNP proliferation in the absence of Shh signaling and does not govern expression of canonical cell cycle genes. Instead, Atoh1 maintains GNPs in a Shh-responsive state by regulating genes that trigger neuronal differentiation, including many expressed in response to bone morphogenic protein-4. Therefore, by targeting multiple genes regulating the differentiation state of GNPs, Atoh1 collaborates with the pro-proliferative Gli1-dependent transcriptional program to influence medulloblastoma development.

Sonic Hedgehog promotes proliferation of Notch-dependent monociliated choroid plexus tumour cells

Aberrant Notch signalling has been linked to many cancers including choroid plexus (CP) tumours, a group of rare and predominantly paediatric brain neoplasms. We developed animal models of CP tumours, by inducing sustained expression of Notch1, that recapitulate properties of human CP tumours with aberrant NOTCH signalling. Whole-transcriptome and functional analyses showed that tumour cell proliferation is associated with Sonic Hedgehog (Shh) in the tumour microenvironment. Unlike CP epithelial cells, which have multiple primary cilia, tumour cells possess a solitary primary cilium as a result of Notch-mediated suppression of multiciliate differentiation. A Shh-driven signalling cascade in the primary cilium occurs in tumour cells but not in epithelial cells. Lineage studies show that CP tumours arise from monociliated progenitors in the roof plate characterized by elevated Notch signalling. Abnormal SHH signalling and distinct ciliogenesis are detected in human CP tumours, suggesting the SHH pathway and cilia differentiation as potential therapeutic avenues.

Elf5 is a principal cell lineage specific transcription factor in the kidney that contributes to Aqp2 and Avpr2 gene expression

The mammalian kidney collecting ducts are critical for water, electrolyte and acid-base homeostasis and develop as a branched network of tubular structures composed of principal cells intermingled with intercalated cells. The intermingled nature of the different collecting duct cell types has made it challenging to identify unique and critical factors that mark and/or regulate the development of the different collecting duct cell lineages. Here we report that the canonical Notch signaling pathway components, RBPJ and Presinilin1 and 2, are involved in patterning the mouse collecting duct cell fates by maintaining a balance between principal cell and intercalated cell fates. The relatively reduced number of principal cells in Notch-signaling-deficient kidneys offered a unique genetic leverage to identify critical principal cell-enriched factors by transcriptional profiling. Elf5, which codes for an ETS transcription factor, is one such gene that is down-regulated in kidneys with Notch-signaling-deficient collecting ducts. Additionally, Elf5 is among the earliest genes up regulated by ectopic expression of activated Notch1 in the developing collecting ducts. In the kidney, Elf5 is first expressed early within developing collecting ducts and remains on in mature principal cells. Lineage tracing of Elf5-expressing cells revealed that they are committed to the principal cell lineage by as early as E16.5. Over-expression of ETS Class IIa transcription factors, including Elf5, Elf3 and Ehf, increase the transcriptional activity of the proximal promoters of Aqp2 and Avpr2 in cultured ureteric duct cell lines. Conditional inactivation of Elf5 in the developing collecting ducts results in a small but significant reduction in the expression levels of Aqp2 and Avpr2 genes. We have identified Elf5 as an early maker of the principal cell lineage that contributes to the expression of principal cell specific genes.

ATOH1 promotes leptomeningeal dissemination and metastasis of Sonic Hedgehog subgroup medulloblastomas

Medulloblastoma arising from the cerebellum is the most common pediatric brain malignancy, with leptomeningeal metastases often present at diagnosis and recurrence associated with poor clinical outcome. In this study, we used mouse medulloblastoma models to explore the relationship of tumor pathophysiology and dysregulated expression of the NOTCH pathway transcription factor ATOH1, which is present in aggressive medulloblastoma subtypes driven by aberrant Sonic Hedgehog/Patched (SHH/PTCH) signaling. In experiments with conditional ATOH1 mouse mutants crossed to Ptch1+/- mice, which develop SHH-driven medulloblastoma, animals with Atoh1 transgene expression developed highly penetrant medulloblastoma at a young age with extensive leptomeningeal disease and metastasis to the spinal cord and brain, resembling xenografts of human SHH medulloblastoma. Metastatic tumors retained abnormal SHH signaling like tumor xenografts. Conversely, ATOH1 expression was detected consistently in recurrent and metastatic SHH medulloblastoma. Chromatin immunoprecipitation sequencing and gene expression profiling identified candidate ATOH1 targets in tumor cells involved in development and tumorigenesis. Among these targets specific to metastatic tumors, there was an enrichment in those implicated in extracellular matrix remodeling activity, cytoskeletal network and interaction with microenvironment, indicating a shift in transcriptomic and epigenomic landscapes during metastasis. Treatment with bone morphogenetic protein or SHH pathway inhibitors decreased tumor cell proliferation and suppressed metastatic tumor growth, respectively. Our work reveals a dynamic ATOH1-driven molecular cascade underlying medulloblastoma metastasis that offers possible therapeutic opportunities. Cancer Res; 77(14); 3766-77. ©2017 AACR.

Abstract B3: Notch 1 signaling-induced choroid plexus tumor arises from epithelial progenitor via Sonic Hedgehog pathway

Introduction: Choroid plexus tumors are rare intraventricular papillary neoplasms of the choroid plexus (CP) epithelium. They occur most often in childhood and comprise 10-20% of all brain tumors in infants. Choroid plexus papilloma is a benign tumor, whereas choroid plexus carcinoma is malignant and most commonly found in pediatric population. Despite good prognosis for choroid plexus papilloma after complete surgical removal, partially resected or inaccessible tumors, and choroid plexus carcinomas are often associated with poor outcomes. Multiple factors including Notch signaling have been implicated in CP tumor development, however, the cell of origin and mechanisms of CP tumor formation remain unclear. In this study, we developed a genetic model of CP tumor induced by Notch pathway activation. Our studies indicate Notch-induced CP tumor is closely related to CP development and is driven by Sonic Hedgehog (Shh) signaling pathway. Methods: Math1-Cre transgenic strain was bred with Rosa26-NICD1 strain that express Notch 1 intracellular domain (NICD1) in a Cre-dependent manner. Math1-Cre/Rosa26-NICD1 animals were further crossed to green fluorescent protein (GFP) Cre reporter (Rosa26-GFP) strain to label Math1-positive lineage from rhombic lip. Transgene expression was analyzed by immunostaining for GFP co-expressed with NICD1 from the Rosa26 locus. Tumor cell proliferation was measured by immunostaining for Ki67 and EdU incorporation assay. Cell differentiation and survival were determined by immunohistochemistry analysis of lineage markers expression and cleaved caspase 3 staining respectively. Gene expression was examined by quantitative RT-PCR analysis and immunostaining. Results: Math1-postive rhombic lip lineage was detected in a small fraction of choroid plexus epithelium of 4th ventricle. The choroid plexus cells derived from Math1+ neural progenitors expressed Lmx1a, Otx2, early markers for CP epithelial lineage, and Aquaporin 1 (AQP1), marker for differentiated CP epithelial cells. In Math1-Cre/Rosa26-NICD1 animals, an abnormal growth was observed in hindbrain CP derived from Math1-positive rhombic lip neural progenitors. EdU incorporation assay and Ki67 staining revealed papillary and intraventricular growth of epithelial cells that exhibited enhanced proliferation compared to control animals in early postnatal period. Though tumor cells expressed Lmx1a, Otx2, they didn9t express AQP1 and MafB, marker for CP mesenchyme. Quantitative RT-PCR analysis revealed that the expression of transthyretin (Ttr), a CP epithelial marker, was reduced in CP tumor cells, while the expression levels of Notch pathway target genes, Hes1 and Hes5, were increased in tumor cells. Proliferating CP tumor cells exhibited higher levels of expression for Gli1, MycN, effectors of the Shh signaling, indicating aberrant activation of Shh pathway. Conclusions: Math1-positive rhombic lip lineage contributes to CP epithelium and is sensitive to Notch 1-induced tumor formation. We have developed a genetic model of CP tumor that exhibits characteristics of pediatric CP tumors. Notch 1-induced CP tumor cells have properties of CP progenitors and lack terminal differentiation. These CP tumors exhibit a transient enhanced proliferation synchronous with enhanced activity of the Shh pathway. Animal models of CP tumors have great potential to facilitate the discovery and development molecular diagnostics and targeted therapies for these rare pediatric malignancies. Citation Format: Li Li, Katie Picotte, Brian Westerhuis, Haotian Zhao. Notch 1 signaling-induced choroid plexus tumor arises from epithelial progenitor via Sonic Hedgehog pathway. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B3.

Abstract 3095: Notch-induced choroid plexus tumor arises from epithelial progenitor and depends on sonic hedgehog signaling for growth

Introduction: Choroid plexus tumors are rare intraventricular papillary neoplasms arising from choroid plexus (CP) epithelium. They occur most often in childhood and comprise 10-20% of all brain tumors in infants. Choroid plexus papilloma is a benign tumor, whereas choroid plexus carcinoma is malignant and most commonly found in pediatric population. Knowledge of the oncogenic processes in CP tumor formation will profoundly impact the development of new and molecular targeted therapies. Previous studies implicate Notch signaling in CP tumorigenesis. However, mechanisms underlying Notch-driven CP tumor formation remain unclear. Animal models of CP tumors have great potential to facilitate the discovery and development of molecular diagnostics and targeted therapies for these rare diseases. Methods and Results: We used the Rosa26-NICD1 strain which will constitutively express the intracellular domain of Notch 1 (NICD1) from the Rosa26 locus after Cre-mediated DNA recombination. The hindbrain CP targeted for NICD1 expression was significantly enlarged with well circumscribed, globular, cauliflower like masses. Histological analysis revealed that CP from NICD1-expressing mutant mice contained an abnormal growth in the 4th ventricle consistent with properties of choroid plexus tumor. Tumor cells exhibit enhanced proliferation transiently after birth: tumor cell proliferation was highest at birth, then gradually decreased and completely stopped at 2 weeks after birth. While CP tumor cells expressed early CP lineage markers such as Lmx1a and Otx2, they didn’t express Aquaporin 1 (AQP1), marker for differentiated CP epithelial cells, suggesting lack of terminal differentiation. We further showed that CP tumor arises during development as ectopic Lmx1a+ cells in CP and exhibit properties similar to those of CP epithelium progenitors. However, unlike progenitor cells, tumor cells fail to exit the cell cycle and differentiate into mature epithelial cells. We characterized changes in gene expression in tumor cells by quantitative RT-PCR. Surprisingly, we detected increased levels of Shh, Gli1 and N-Myc, effectors of the Sonic Hedgehog (Shh) pathway, in proliferating tumor cells. CP tumor cells cultured in serum-free conditions underwent rapid proliferation in the presence of Shh. Ultra-structural studies revealed lack of multi-ciliation differentiation in tumors cells, whereas the expression of Foxj1 and Midas, crucial regulators of cilia differentiation, was significantly downregulated. Conclusions: We developed a genetic model of CP tumor driven by Notch signaling activation. Notch 1-induced CP tumor arises during development from CP epithelial progenitors but lack terminal differentiation and multi-ciliation of mature CP epithelial cells. The proliferation of Notch 1-induced tumors is supported by Shh pathway, indicating Shh pathway as a therapeutic target for Notch-driven CP tumors. Citation Format: Haotian Zhao, Li Li, Katie Picotte. Notch-induced choroid plexus tumor arises from epithelial progenitor and depends on sonic hedgehog signaling for growth. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3095. doi:10.1158/1538-7445.AM2014-3095